Molecular identification and sequence analysis of Clostridium perfringens virulence genes isolated from sheep and goats
Published:
Apr 18, 2023
Updated:
2023-04-18
Versions:
2023-04-18 (2)
Keywords:
Clostridium perfringens Genetic diversity Sequence Toxin type Virulence
Abstract
Clostridium perfringens (C. perfringens) belongs to the family of Clostridiaceae and produces a wide range of toxins (four major and a variety of minor toxins). Some toxins associated with virulence have been shown to participate in the pathogenesis of enteric diseases in sheep, goats, and other animals. The aim of this study was to determine the presence of minor virulence genes and their genetic diversity in the various toxinotypes of C. perfringens isolates. About 84 isolates collected from sheep and goat flocks were examined and sequenced for the presence of minor virulence genes. Results showed that PFO and cpb2 were found in 79 out of 84 (94%), cpe was identified in 29 out of 84 (35%) and the presence of tpeL was confirmed in 28 out of 84 (33%) isolates, while none of the isolates were identified as carrying the netB gene. This study shows that the prevalence of genes varied among various types of C. perfringens isolates and also sheep and goat samples, furthermore these findings change the toxinotypes of isolates based on a modified scheme of toxinotype, which incorporated CPE and NetB toxins. The results of this study showed that the dominant minor virulence genes were PFO and cpb2 and the occurrence of cpe and tpeL genes was also diverse. DNA sequencing revealed approximately a sequence similarity of 97-100% with the GenBank database and 3 motation was found in sequence analysis.
Article Details
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Amini, M., Kheirkhah, B., Shamsaddini Bafti, M., & Rokhbakhsh Zamin, F. (2023). Molecular identification and sequence analysis of Clostridium perfringens virulence genes isolated from sheep and goats. Journal of the Hellenic Veterinary Medical Society, 74(1), 5345–5354. https://doi.org/10.12681/jhvms.29434 (Original work published April 11, 2023)
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- Vol. 74 No. 1 (2023)
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References
Ahsani, M., Bafti, M.S., Esmailizadeh, A. and Mohammadabadi, M.
(2011) Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep. Small. Rumin. Res. 95(1):65-69.
Ahsani, M., Mohammadabadi, M. and Shamsaddini, M. (2010) Clostridium perfringens isolate typing by multiplex PCR. J. Venom Anim.
Toxin Incl. Trop. Dis. J. 16(4): 573-578.
Allaart, J.G., van Asten, A.J. and Gröne, A. (2013) Predisposing factors
and prevention of Clostridium perfringens-associated enteritis.com.
Immunol. Microbiol. Infect. Dis. 36(5): 449-464.
Amimoto, K., Noro, T., Oishi, E. and Shimizu, M. (2007) A novel toxin
homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C. Microbiol. 153(4): 1198-
Athira, C.K., Milton, A.A.P., Reddy, A., Rajendrakumar, A.M., Verma,
M.R., Kumar, A., Nagaleekar, V.K. and Agarwal, R.K. (2018) Diversity of toxin-genotypes among Clostridium perfringens isolated from
healthy and diarrheic neonatal cattle and buffalo calves. Anaerobe 49:
-102.
Brito, C., Mesquita, F.S., Bleck, C.K., Sellers, J.R., Cabanes, D. and
Sousa, S. (2019) Perfringolysin o-induced plasma membrane pores
trigger actomyosin remodeling and endoplasmic reticulum redistribution. Toxins 11(7): 419.
Chen, J. and McClane, B.A. (2015) Characterization of Clostridium perfringens TpeL toxin gene carriage, production, cytotoxic contributions, and trypsin sensitivity. Infect. Immun. 83(6): 2369-2381.
Michel R and Stiles, B.G. (2005) 17 Clostridial Toxins Vs. Other Bacterial Toxins. Handbook on clostridia, 323.
Deguchi, A., Miyamoto, K., Kuwahara, T., Miki, Y., Kaneko, I., Li, J.,
McClane, B.A. and Akimoto, S. (2009) Genetic characterization of
type A enterotoxigenic Clostridium perfringens strains. PLoS One
(5): e5598.
Duracova, M., Klimentova, J., Myslivcova Fucikova, A., Zidkova, L.,
Sheshko, V., Rehulkova, H., Dresler, J. and Krocova, Z. (2019) Targeted mass spectrometry analysis of Clostridium perfringens toxins.
Toxins 11(3): 177.
Ezatkhah, M., Alimolaei, M., Amini, M. and Shamsaddini Bafti, M.
(2016) Typing toxigenic Clostridium perfringens strains from the ruminants of Yazd province by multiplex polymerase chain reaction.
Int. J. Enteric Pathog. 4(3):1-4.
Yadegar F., Nakhaeie P., Hashemtabar G., Kalidari Gh., Rashtibaf M. and
Razmyar J. (2018) Major and minor toxins of Clostridium perfringens isolated from healthy and diseased sheep. Small. Rumin. Res. 168:1-5.
Fayez, M., Elsohaby, I., Al-Marri, T., Zidan, K., Aldoweriej, A., El-Sergany, E. and Elmoslemany, A. (2020) Genotyping and antimicrobial
susceptibility of Clostridium perfringens isolated from dromedary
camels, pastures and herders. Comp. Immunol, Microbiol. Infect.
Dis. 70: 101460.
Fernandez-Miyakawa, M.E., Jost, B.H., Billington, S.J. and Uzal, F.A.
(2008) Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model. Vet.
Microbiol. 127(3-4): 379-385.
Forti, K., Ferroni, L., Pellegrini, M., Cruciani, D., De Giuseppe, A., Crotti,
S., Papa, P., Maresca, C., Severi, G. and Marenzoni, M.L. (2020) Molecular characterization of Clostridium perfringens strains isolated in
Italy. Toxins 12(10): 650.
Freedman, J.C., Shrestha, A. and McClane, B.A. (2016) Clostridium perfringens enterotoxin: action, genetics, and translational applications.
Toxins 8(3): 73.
Greco, G., Madio, A., Buonavoglia, D., Totaro, M., Corrente, M., Martella, V. and Buonavoglia, C. (2005) Clostridium perfringens toxin-types in lambs and kids affected with gastroenteric pathologies in
Italy. The Vet. J. 170(3): 346-350.
Gurjar, A., Li, J. and McClane, B.A. (2010) Characterization of toxin
plasmids in Clostridium perfringens type C isolates. Infect. Immun.
(11): 4860-4869.
Heikinheimo, A. and Korkeala, H. (2005) Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish
broiler chickens. Lett. Appl. Microbiol. 40(6): 407-411.
Jabbari, A., Tekyei, F., Esmaelizad, M. and Pilechian, L.R. (2012) Occurrence of Beta2 toxigenicClostridium perfringens isolates with different toxin types in Iran. Arch. Razi 67:133-137.
Keyburn, A.L., Boyce, J.D., Vaz, P., Bannam, T.L., Ford, M.E., Parker, D.,
Di Rubbo, A., Rood, J.I. and Moore, R.J. (2008) NetB, a new toxin
that is associated with avian necrotic enteritis caused by Clostridium
perfringens. PLoS Pathog. 4(2): e26.
Keyburn, A.L., Portela, R.W., Sproat, K., Ford, M.E., Bannam, T.L., Yan,
X., Rood, J.I. and Moore, R.J. (2013) Vaccination with recombinant
NetB toxin partially protects broiler chickens from necrotic enteritis.
Vet. Res. 44(1), 1-8.
Kiu, R. and Hall, L.J. (2018) An update on the human and animal enteric
pathogen Clostridium perfringens. Emerg.Microb. Infect 7(1): 1-15.
Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018) MEGA
X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology, Evolution 35(6): 1547.
Li, J., Adams, V., Bannam, T.L., Miyamoto, K., Garcia, J.P., Uzal, F.A.,
Rood, J.I. and McClane, B.A. (2013) Toxin plasmids of Clostridium
perfringens. Microbiol. Mol. Biol. Review 77(2):208-233.
Martin, T.G. and Smyth, J.A. (2009) Prevalence of netB among some clinical isolates of Clostridium perfringens from animals in the United
States. Vet. Microbiol. 136(1-2):202-205.
Matsuda, A., Aung, M.S., Urushibara, N., Kawaguchiya, M., Sumi, A.,
Nakamura, M., Horino, Y., Ito, M., Habadera, S. and Kobayashi, N.
(2019) Prevalence and genetic diversity of toxin genes in clinical isolates of Clostridium perfringens: Coexistence of alpha-toxin variant
and binary enterotoxin genes (bec/cpile). Toxins 11(6); 326.
Mehdizadeh Gohari, I., A. Navarro, M., Li, J., Shrestha, A., Uzal, F. and
A. McClane, B. (2021) Pathogenicity and virulence of Clostridium
perfringens. Virulence 12(1): 723-753.
Mignaqui, A.C., Marcellino, R., Ronco, T., Pappalardo, J.S., Nonnemann,
B., Pedersen, K. and Robles, C.A. (2017) Isolation and molecular
characterization of Clostridium perfringens from healthy Merino
lambs in Patagonia region, Argentina. Anaerobe 43: 35-38.
Mohiuddin, M., Iqbal, Z., Siddique, A., Liao, S., Salamat, M.K.F., Qi, N.,
Din, A.M. and Sun, M. (2020) Prevalence, Genotypic and Phenotypic Characterization and Antibiotic Resistance Profile of Clostridium
perfringens Type A and D Isolated from Feces of Sheep (Ovis aries)
and Goats (Capra hircus) in Punjab, Pakistan. Toxins 12(10): 657.
Navarro, M.A., McClane, B.A. and Uzal, F.A. (2018) Mechanisms of action and cell death associated with Clostridium perfringens toxins.
Toxins 10(5): 212.
Park, M. and Rafii, F. (2019) The prevalence of plasmid-coded cpe enterotoxin, β2 toxin, tpeL toxin, and tetracycline resistance in Clostridium perfringens strains isolated from different sources. Anaerobe
, 124-129.
Revitt-Mills, S.A., Rood, J.I. and Adams, V. (2015) Clostridium perfringens extracellular toxins and enzymes: 20 and counting. Microbiol.
Aust. 36(3): 114-117.
Rood, J.I., Adams, V., Lacey, J., Lyras, D., McClane, B.A., Melville, S.B.,
Moore, R.J., Popoff, M.R., Sarker, M.R. and Songer, J.G. (2018) Expansion of the Clostridium perfringens toxin-based typing scheme.
Anaerobe 53: 5-10.
Rood, J.I., Keyburn, A.L. and Moore, R.J. (2016) NetB and necrotic enteritis: the hole movable story. Avian Path. 45(3): 295-301.
Sayeed, S., Li, J. and McClane, B.A. (2010) Characterization of virulence
plasmid diversity among Clostridium perfringens type B isolates. Infect. Immun. 78(1): 495-504.
Sharpe, D. (2015) Chi-square test is statistically significant: Now what?.
Pract. Assessment, Research, and Evaluation. 20(1):8.
Singh, D.D., Pawaiya, R.S., Gururaj, K., Gangwar, N.K., Mishra, A.K.,
Andani, D., Singh, M.K., Bhushan, S. and Kumar, A. (2018) Molecular detection of Clostridium perfringens toxinotypes, Enteropathogenic Escherichia coli, rotavirus and coronavirus in diarrheic fecal samples of neonatal goat kids. Veterinarski Arhiv 88(1): 1-20.
Songer, J.G. and Uzal, F.A. (2005) Clostridial enteric infections in pigs. J.
vet. Diagn. Invest. 17(6); 528-536.
Uzal, F., Vidal, J., McClane, B. and Gurjar, A. (2010) Clostridium perfringens toxins involved in mammalian veterinary diseases. The open
Toxinology J. 2: 24.
Uzal, F.A., Freedman, J.C., Shrestha, A., Theoret, J.R., Garcia, J., Awad,
M.M., Adams, V., Moore, R.J., Rood, J.I. and McClane, B.A. (2014)
Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol. 9(3): 361-377.
Uzal, F.A. and Songer, J.G. (2008) Diagnosis of Clostridium perfringens
intestinal infections in sheep and goats. J. Vet. Diagn. Invest. 20(3):
-265.
Van Asten, A.J., Nikolaou, G.N. and Gröne, A. (2010) The occurrence
of cpb2-toxigenic Clostridium perfringens and the possible role of
the β2-toxin in enteric disease of domestic animals, wild animals and
humans. Vet. J. 183(2): 135-140.
Verherstraeten, S., Goossens, E., Valgaeren, B., Pardon, B., Timbermont, L., Haesebrouck, F., Ducatelle, R., Deprez, P., Wade, K.R. and
Tweten, R. (2015) Perfringolysin O: the underrated Clostridium perfringens toxin, Toxins 7(5): 1702-1721.
Woudstra, C., Le Maréchal, C., Souillard, R., Anniballi, F., Auricchio, B.,
Bano, L., Bayon-Auboyer, M.-H., Koene, M., Mermoud, I. and Brito,
R.B. (2018) Investigation of Clostridium botulinum group III’s mobilome content. Anaerobe 49:71-77.
Zaragoza, N.E., Orellana, C.A., Moonen, G.A., Moutafis, G. and Marcellin, E. (2019) Vaccine production to protect animals against pathogenic clostridia. Toxins 11(9): 525
